Bicyclic tetrahydroxylated pyrrolizidines

ABSTRACT

Novel bicyclic tetrahydroxylated pyrrolizidines are disclosed which are inhibitors of glycosidase enzymes. A preferred inhibitor is 1α, 2α, 6α, 7α, 7αβ-1,2,6,7-tetrahydroxypyrrolizidine. It is synthesized from D-glycero-D-gulo-heptono-1,4-lactone. 
     Novel Intermediate compounds prepared during this synthesis are 7-O-tert-butyldiphenylsilyl-2,3:5,6-di-O-isopropylidene-D-glycero-D-gulo-hept ono-1,4-lactone and 1α, 2α, 6α, 7α, 7αβ-1,2:6,7-di-O-isopropylidene-1,2,6,7-tetrahydroxypyrrolizidine.

BACKGROUND OF THE INVENTION

This invention relates to novel bicyclic tetrahydroxylatedpyrrolizidines and methods for their chemical synthesis. These compoundsare useful inhibitors of glycosidase enzymes.

Several naturally occurring polyhydroxylated pyrrolidines,pyrrolizidines and indolizidines are powerful and specific inhibitors ofglycosidases [Fellows and Fleet, Alkaloidal Glycosidase Inhibitors fromPlants, in Natural Products Isolation (Ed. G. H. Wagman and R. Cooper),Elsevier, Amsterdam, 1988, pp. 540-560; Evans et al, Phytochemistry, 241953-1956 (1985)]. In recent years, plagiarism of plant chemistry hasled to the synthesis of powerful inhibitors of other glycosidases [Fleetet al,, J. Chem. Soc., Perkin Trans. 1, 665-666, (1989); Bashyal et al,Tetrahedron 43, 3083-3093 (1987), and Fleet et al, Tetrahedron 43,979-990 (1987)]. It is now clear that, although changes instereochemistry of the hydroxyl groups have profound effects on theselectivity of glycosidase inhibition, it is not easy to predict theeffects of such changes [Fleet et al, Tetrahedron Lett., 26 3127-3131(1985)]. For example, 6-episcastanospermine (2) is a glucosidaseinhibitor even though the stereochemistry of the four adjacent chiralcentres in the piperidine is similar to those in the pyranose form ofmannose [Molyneux et al, Arch. Biochem. Biophys., 251, 450-457 (1986)].Similarly, 1,7a-diepialexine (3), structurally very similar to thepowerful mannosidase inhibitor swainsonine (4), is an inhibitor offungal glucan 1,4-α-glucosidase [Nash et al, Phytochemistry, submittedfor publication]. Also, β-C-methyl deoxymannojirimycin (5) is a strongand specific α-L-fucosidase inhibitor and has no effect on human liverα-mannosidase [Fleet et al, Tetrahedron Lett., 30, In Press (1989)].##STR1##

With a few exceptions [Raymond and Vogel, Tetrahedron Lett., 30 705-706(1989)], sugars have been the starting materials used in the synthesisof such compounds as castanospermines [such as (2)], Setoi et al,Tetrahedron Lett., 26 4617-4620 (1985), Hamana et al, J. Org. Chem., 52,5492-5494 (1987) and Fleet et al, Tetrahedron Lett., 29, 3603-3606(1988); alexines [such as (3)], Fleet et al, Tetrahedron Lett., 29,5441-5445 (1988); and homonojirimycins [such as (4)], Anzeneno et al, J.Org. Chem. 54, 2539-2542 (1989). Invariably in the syntheses of thesecompounds with five adjacent chiral centres and six or seven adjacentfunctional groups, the strategy chosen has been to start from a hexoseand to introduce the additional chiral centre late in the synthesis. Analternative is to start from derivatives of heptoses, that is by veryearly introduction of the additional chiral centre.

Relatively few studies have been reported on the protecting groupchemistry of even readily available heptonolactones [Brimacombe andTucker, Carbohydr. Res. 2, 341-348 (1966)]. Likewise, only a fewexamples of syntheses from heptose derivatives have been reported. Oneneat example is described by Stork et al, J. Am. Chem. Soc. 100,8272-8273 (1978). Recently, a research group led by co-inventor Fleetherein has found that suitably protected heptonolactones can be powerfuland readily manipulatable chiral pool materials. See Bruce et al,Tetrahedron 45, In press 1989, and copending application Ser. No.07/352,068, filed May 15, 1989.

BRIEF DESCRIPTION OF THE INVENTION

In accordance with the present invention, novel bicyclictetrahydroxylated pyrrolizidines are synthesized from the readilyavailable heptonolactones, D-glycero-D-gulo-heptono-1,4-lactone and theanalogous D-glycero-D-talo-heptono-1,4-lactone.

In a preferred embodiment of the invention, the novel 1α, 2α, 6α, 7α,7α, β-1,2,6,7-tetrahydroxypyrrolizidine (1) is prepared fromD-glycero-D-gulo-heptono-1,4-lactone by two different synthetic routes.This novel tetrahydroxylated pyrrolizidine is an effective inhibitor ofhuman liver glycosidases. ##STR2##

The tetrahydroxyprrolizidone (1) is an analogue of 1,8-diepiswainsonine.A similar analogue of swainsonine can be made by analogous methodsstarting with D-glycero-D-talo-heptono-1,4-lactone to produce the novel(1S, 2R, 6R, 7S)-1,2,6,7-tetrahydroxypyrrolizidine.

DETAILED DESCRIPTION OF THE INVENTION

The invention is conveniently illustrated by the following descriptionof the preferred embodiments in which 1α, 2α, 6α, 7α,7αβ-1,2,6,7-tetrahydroxypyrrolizidine (1) is synthesized fromD-glycero-D-gulo-heptono-1,4-lactone (6) by two different Methods A andB, in ten steps as follows in which compound numbers in parenthesescorrespond to compounds shown by chemical structure herein:

A.

(1) The primary hydroxyl group in heptonolactone (6) is treated with asilyl blocking agent such as tert-butyldiphenylsilyl chloride to givethe protected lactone (7).

(2) The protected lactone (7) is reacted with 2,2-dimethoxypropane toprovide the fully protected lactone or diacetonide (9).

(3) The diacetonide (9) is reacted with fluoride ion to cleave the silylether at C7 and thereby provide access to nitrogen in the ring and givethe primary alcohol (10).

(4) The primary alcohol (10) is esterified with triflic anhydride toafford the triflate (11).

(5) The triflate (11) is reacted with azide ion to give the azidolactone(12).

(6) The azidolactone (12) is reduced to the azidodiol (13).

(7) The azidodiol (13) is reacted with methanesulfonyl chloride toprovide the azidodimesylate (14).

(8) The azidodimesylate (14) is catalytically hydrogenated in ethanol atambient temperature.

(9) The product from step 8 is heated in ethanol in the presence ofsodium acetate to give the tetracyclic pyrrolizidine (15).

(10) The acetonide groups in the tetracyclic pyrrolizidine (15) areremoved by acid hydrolysis to give the product 1α, 2α, 6α, 7α,7αβ-1,2,6,7-tetrahydroxypyrrolizidine (1). ##STR3##

B.

Steps 1 and 2 are the same as in Method A.

(3) The fully protected lactone or diacetonide (9) is reduced to givethe silyl diol (16).

(4) The silyl diol (16) is reacted with methanesulfonyl chloride toprovide the dimesylate (17).

(5) Nitrogen is introduced into the ring by reaction of the dimesylate(17) with benzylamine to give the monocylic pyrrolidine (20).

(6) The silyl protecting group is removed from C7 of the monocylicpyrrolidine (20) by treatment with fluoride ion to provide the primarydiol (21).

(7) The primary diol (21) is reacted with methanesulfonyl chloride togive the unstable mesylate (22) which spontaneously closes to form thesecond pyrrolidine ring and give the N-benzyl pyrrolizidinium salt (23).

(8) The N-benzyl group in (23) is cleaved by catalyzed hydrogenation.

(9) Neutralization of the product of step 8 gives the tetracyclicpyrrolizidine (15).

(10) The acetonide groups in the tetracyclic pyrrolizidine (15) areremoved by acid hydrolysis to give the product 1α, 2α, 6α, 7α,7αβ-1,2,6,7-tetrahydroxypyrrolizidine (1). ##STR4##

The fully protected lactone or diacetonide (9), namely7-O-tert-butyldiphenylsilyl-2,3:5,6-di-O-isopropylidene-D-glycero-D-gulo-heptono-1,4-lactone,is a novel intermediate that can be used as a starting material for eachof Methods A and B, above. Both Methods A and B result in preparation ofthe novel fully protected tetracyclic pyrrolizidine (15), namely 1α-2α,7α, 7αβ-1,2,6,7-di-O-isopropylidene-1,2,6,7-tetrahydroxy pyrrolizidine,from which the protecting groups can readily be removed by acidhydrolysis.

Other such suitable reactants for use in the foregoing syntheses ofMethods A and B will be apparent to the person skilled in the art afterreading the present disclosure. These reactants are generally used inproportions such as to satisfy the stoichiometry of the above reactionsteps. Illustrative of such other reactants are the use oft-butyldimethylsilyl chloride to introduce the silyl protecting groups;use of other ketones, e.g., acetone, 3-pentanone, dihexylketone,cyclohexanone, and the like to introduce suitable hydroxyl protectinggroups; use of other azide cations to introduce the azide group, e.g.potassium, lithium and tetra-butylammonium; and use of other solventmedia such as DMF, THF, DMSO, N-methylpyrrolidine, acetonitrile and thelike.

The foregoing reactions in Methods A and B were illustratively carriedout as follows:

SYNTHESIS OF TETRAHYDROXYPYRROLIZIDINE (1)

A. The synthesis of 1α, 2α, 6α, 7αβ-1,2,6,7-tetrahydroxypyrrolizidine(1), with five adjacent chiral centres and seven adjacent carbon atomsbearing functional groups, requires the joining of C-1, C-4 and C-7 ofthe heptonolactone (6) by nitrogen with inversion of configuration atC-4. The order in which the formation of the different carbon-nitrogenbonds are formed is variable, although protection of the hydroxyl groupsat C-2, C-3, C-5 and C-6 is required; bis-isopropylidene protection ofthe hydroxyl functions assists the intramolecular cyclizations to thepyrrolidine rings, since fused five-five membered rings are formed.

The primary hydroxyl group in (6) was protected as thetert-butyldiphenylsilyl ether by reaction with tert-butyldiphenylsilylchloride in the presence of imidazole to afford (7) in 55% yield[Hanessian and Lavallee, Can. J. Chem. 53, 2975-2977 (1975)]. Althoughthe silyl chloride was present in only slight excess, a significantamount (18%) of a disilyl derivative was also formed; the structure ofthis by-product was tentatively assigned as the 2,7-disilylether (8),since hydroxyl groups α- to lactone carbonyl groups show enhancedreactivity in silylation reactions [Mark and Zbiral, Monatsch. Chem.112, 215-239 (1981)]. Reaction with 2,2-dimethoxypropane in the presenceof a catalyst of dl-camphor sulphonic acid gave the diacetonide (9) [68%yield], in which the presence of two 5-ring ketals is clearly indicatedby two singlets for the quaternary isopropylidene carbons at about δ110in the ¹³ C NMR spectrum; the quaternary carbon of a six ring ketalgenerally appears below δ100. If the acetonation reaction was stoppedbefore completion, both 5- and 6-ring monoacetonides could be isolatedfrom the reaction mixture, indicating that (9) is the thermodynamicproduct.

One approach to the synthesis of (1) from the divergent intermediate (9)requires initial introduction of nitrogen at C-7. Access was gained toC-7 by cleavage of the silyl ether with fluoride ion to give the primaryalcohol (10) in 86% yield. Esterification of (10) with trifluoromethanesulphonic anhydride afforded the triflate (11) which with sodium azidein dimethylformamide at room temperature gave the azide (12) [77% yieldfrom (10)]. The lactone (12) was reduced by sodium borohydride inethanol to the azidodiol (13) [93% yield] which was reacted with excessmethanesulphonyl chloride in pyridine in the presence of4-dimethylaminopyridine to give the dimesylate (14) [94% yield].Hydrogenation of the azidodimesylate (14) in ethanol in the presence ofa catalyst of palladium black, followed by heating in ethanol in thepresence of sodium acetate, lead directly to the tetracyclicpyrrolizidine (15) in 76% yield. In (15), C-1 is equivalent with C-7,C-2 with C-6 and C-3 with C-5 giving only five signals in the δ2.5-5.0region of the ¹ H NMR sprectrum, and only four signals in the δ55-85region of the ¹³ C NMR spectrum; additionally in the .sup. 13 C NMRspectrum, the quaternary isopropylidene carbons are equivalent and thereare two pairs of equivalent isopropylidene methyl carbons. Removal ofthe acetonide groups from (15) by treatment with aqueous trifluoroaceticacid gave the desired tetrahydroxypyrrolizidine (1) in 90% yield [15%overall yield for the ten steps from heptonolactone (6)]. It is clearthat removal of the two cyclic ketals in (15) has resulted in a changeof the torsion angles within the structure, since there are significantchanges in the coupling constants between (1) and (15).

B. An alternative synthesis of (1) from the fully protected lactone (9)involves initial formation of a pyrrolidine ring between C-1 and C-4.Reduction of the lactone (9) with lithium aluminum hydride intetrahydrofuran gave the diol (16) in the 77% yield, providing access tothe C-1 and C-4 hydroxyl groups while all the other oxygen functions areprotected. The silyl diol (16) was then converted into the dimesylate(17) [66% yield] by treatment with methanesulphonyl chloride in pyridinein the presence of 4-dimethylaminopyridine; the anhydrosugar (19) [32%yield] was also obtained in this reaction, presumably arising fromintramolecular cyclization of the monomesylate (18). Nitrogen wasintroduced by reaction of the dimesylate (17) with benzylamine givingthe monocyclic pyrrolidine (20) in 72% yield; efficient cyclization of1,4-dimesylates to pyrrolidines on treatment with benzylamine has beenreported by Fleet et al., Tetrahedron44, 2469-2655 (1988); Fleet andSon, Ibid. 44, 2637-2647 (1988). The formation of the second pyrrolidinering was achieved by first removing the silyl protecting group from C-7of (20) by treatment with fluoride ion (84% yield). Subsequentmesylation of the primary alcohol (21) gave the unstable mesylate (22)which spontaneously closed to give the N-benzyl pyrrolizidinium salt(23). Cleavage of the N-benzyl group by hydrogenation of (23) in ethanolin the presence of palladium black, followed by neutralization withsodium bicarbonate gave the pyrrolizidine diacetonide (15) [31% yieldfrom (21)], identical in all respects to the sample of (15) prepared bythe alternative Method A, above.

GLYCOSIDASE INHIBITION

The effect of 1α,2α,6α,7α,7αβ-1,2,6,7-tetrahydroxypyrrolizidine (1) onthe activity of 12 human liver glycosidases was tested by assay methodsdescribed by Daher et al., Biochem. J.258, 613-615 (1989). The compound(1) is a weak inhibitor of all human lysosomal, Golgi II and neutralα-mannosidases (I₅₀ approximately 1 mM); in addition it is also a weakinhibitor of α-fucosidase, α-and β-galactosidase, and the broadspecificity β-galactosidase/β-glucosidase. The pyrrolizidine (1) isstructurally related to 1,4-dideoxy-1,4-imino-L-allitol (DIA) (24) whichis also a relatively weak inhibitor of lysosomal α-mannosidase (K_(i)1.2×10⁻⁴ M). DIA (24) is comparable to the pyrrolizidine (1) in itsinhibition of the neutral and Golgi II α-mannosidases [Cenci di Bello etal., Biochem. J.259, 855-861 (1989)]; both DIA and (1) have a relativelybroad specificity of inhibition of glycosidases [Daher et al., supra.].In contrast, the closely related indolizidine 8,8a-diepiswainsonine (25)is a very effective inhibitor of lysosomal (K_(i) 2×10⁻⁶ M) and Golgiprocessing α-mannosidase, both in vivo and in vitro, and theindolizidine (25) fits the active site of the α-mannosidases moreclosely than (1) or (24).

The following examples will further illustrate the invention in greaterdetail although it will be appreciated that the invention is not limitedto these specific examples.

METHODS

Melting points were recorded on a Kofler block and are corrected.Infrared spectra were recorded on either a Perkin-Elmer 781spectrophotomer or a Perkin-Elmer 1750 IR FT spectrometer. Opticalrotations were measured on a Perkin-Elmer 241 polarimeter with apath-length of 10 cm; concentrations are given in g/100 ml. ¹ H NMRspectra were run either at 200 MHz on a Varian Gemini 200 spectrometer,or at 300 MHz on a Bruker WH 300 spectrometer. Chemical shifts arequoted on the scale using residual solvent as an internal standard. ¹³ CNMR spectra were recorded at 50 MHz on a Varian Gemini 200 spectrometer;for samples in D₂ O, dioxan (δ67.2) was added as a reference. Massspectra were recorded on either a VG Micromass ZAB 1F, a VG Mass 1ab20-250 or a TRIO 1 spectrometer using chemical ionization (CI) ordesorption chemical ionization (DCI) techniques. Microanalyses wereperformed by the microanalytical service of the Dyson PerrinsLaboratory, Oxford, U.K. T.l.c. was performed on glass plates coatedwith silica gel Blend 41 (80% silica gel HF₂₅₄ and 20% silica gel G) oron aluminum plates coated with Merck silica gel 60F₂₅₄. Compounds werevisualized with a spray of 0.2% w/v ceric sulphate and 5% ammoniummolybdate in 2 M sulphuric acid, or 0.5% ninhydrin in methanol (foramines). Flash chromatography was carried out using Sorbsil C60 40/60flash silica gel. Dry column chromatography was carried out using MerckKieselgel 60H. Ion exchange columns were packed with Aldrich 50X, 8-100resin in the H⁺ form. Pyridine and benzylamine were distilled (andstored) over potassium hydroxide. Hexane was distilled to removeinvolatile fractions. Immediately prior to use, dimethylformamide (DMF)and dichloromethane were distilled from calcium hydride, andtetrahydrofurane (THF) was distilled from sodium benzophenone ketyl.D-glycero-D-gulo-Heptono-1,4-lactone (6) was obtained from Sigma.

EXAMPLE 17-O-tert-Butyldiphenylsilyl-D-glycero-D-gulo-heptono-1,4-lactone (7) and2,7-di-O-tert-Butyldiphenylsilyl-D-glycero-D-gulo-heptono-1,4-lactone(8)

D-glycero-D-gulo-Heptono-1,4-lactone (6) (10 g, 48.08 mmol) andimidazole (4.98 g, 1.5 equiv) were added to dry DMF (25 ml) and themixture stirred at 0° C. under nitrogen. tert-Butylchlorodiphenylsilane(13.74 ml, 1.1 equiv) was added slowly, after which the reaction mixturewas allowed to warm up to room temperature over three hours. After 22hours, t.l.c. (eluant ethyl acetate) indicated that the mixturecontained the desired monosilyl derivative (R_(f) 0.65) and a smalleramount of another carbohydrate derivative (R_(f) 0.9). The crudereaction mixture was shaken with water (50 ml), causing a whiteprecipitate to form. Ethyl acetate (90 ml) was added and the layersseparated after shaking. The aqueous layer was back-extracted with moreethyl acetate (25 ml). The combined organic extracts were washed withsaturated aqueous sodium chloride (4×25 ml) and dried (magnesiumsulphate). Evaporation of the solvent followed by dry columnchromatography (eluant hexane:ethyl acetate, 2:1, increasing the eluantpolarity with each fraction), yielding 7-O-tert-butyldiphenylsilyl-D-glycero-D-gulo-heptono-1,4-lactone (7)(11.02 g, 55%) as a white solid, m.p. 54°-57° C. (Found: C, 61.58; H,6.86%. C₂₃ H₃₀ O₇ Si requires: C, 61.87; H, 6.77%); [α]_(d) ²⁰ -10.56°(c, 0.99 in CHCl₃); v_(max) (CHCl₃) 3410 (broad, OH) and 1790 cm⁻¹(γ-lactone); and2,7-di-O-tert-butyldiphenylsilyl-D-glycero-D-gulo-heptono-1,4-lactone(8) (5.94 g, 18%) as a colorless, viscous oil [α]_(D) ²⁰ -4.08° (c, 1.20in CHCl₃); v_(max) (CHCl₃) 3440 (broad, OH) and 1790 cm⁻¹ (γ-lactone).

EXAMPLE 27-O-tert-Butyldiphenylsilyl-2,3:5,6-di-O-isopropylidene-D-glycero-D-gulo-heptono-1,4-lactone(9)

7-O-tert-Butyldiphenylsilyl-d-glycero-D-gulo-heptono-1,4-lactone (7)(3.00 g, 6.73 mmol) and d1-camphor sulphonic acid (0.15 g, 5%) weredissolved in dry acetone (60 ml). 2,2-Dimethoxypropane (3.50 g, 5 equiv)was added and the mixture was stirred at 50° C. under reflux for 22hours. The reaction was quenched by addition of excess sodium hydrogencarbonate, at which stage t.l.c. (eluant hexane:ethyl acetate, 6:1)indicated that the reaction mixture contained three compounds, one majorproduct (R_(f) 0.6) together with two minor products (R_(f) 0.8 and0.1). After filtration and evaporation of the solvent, the residue waspurified by flash chromatography (eluant hexane:ethyl acetate, 8:1),yielding7-O-tert-butyldiphenylsilyl-2,3:5,6-di-O-isopropylidene-D-glycero-D-gulo-heptono-1,4-lactone(9) (2.40 g, 68%) as a white, crystalline solid, m.p. 104°-106° C.(Found: C, 66.19; H, 7.58%. C₂₉ H₃₈ O₇ Si requires: C, 66.13; H, 7.27%);[α] _(D) ²⁰ -21.64° (c, 0.98 in CHCl₃); v_(max) (CHCl₃) 1790(γ-lactone), 1386 and 1377 cm⁻¹ (CMe₂).

EXAMPLE 32,3:5,6-Di-O-isopropylidene-D-glycero-D-gulo-heptono-1,4-lactone (10)

7-O-tert-Butyldiphenylsilyl-2,3:5,6-di-O-isopropylidene-D-glycero-D-gulo-heptono-1,4-lactone(9) (4.11 g, 7.81 mmol) was dissolved in dry THF (200 ml) and thesolution was stirred under nitrogen. Tetra-n-butylammonium fluoride(11.7 ml of a 1M solution in THF, 1.5 equiv) was added dropwise. Afterone and a half hours t.l.c. (eluant hexane:ethyl acetate, 6:1) indicatedone product at the baseline but no starting material (R_(f) 0.6).Evaporation of the solvent gave a pale yellow oil which was purified byflash chromatography (eluant ethyl acetate:hexane, 3:2) yielding2,3:5,6-di-O-isopropylidene-D-glycero-D-gulo-heptono-1,4-lactone (10)(1.93 g, 86%) as a white, crystalline solid, m.p. 115°-120° C. (Found:C, 54.46; H, 6.99%. C₁₃ H₂₀ O₇ requires: C, 54.16; H, 6.99%); [α]_(D) ²⁰-53.40° (c, 1.05 in CHCl₃); v_(max) (CHCl₃) 3560 (OH), 1790 (γ-lactone),1388 and 1379 cm⁻¹ (CMe₂).

EXAMPLE 47-Azido-7-deoxy-2,3:5,6-di-O-isopropylidene-D-glycero-D-gulo-heptono-1,4-lactone(12)

2,3:5,6-Di-O-isopropylidene-D-glycero-D-gulo-heptono-1,4-lactone (10)(0.50 g, 1.74 mmol) was dissolved in dry dichloromethane (50 ml) and drypyridine (0.28 ml, 2 equiv) was added and the solution was stirred at-30° C. under nitrogen. Trifluoromethanesulphonic anhydride (0.44 ml,1.5 equiv) was added slowly, and after 30 minutes, t.l.c. (eluant ethylacetate:hexane, 2:1) indicated complete conversion to product (R_(f)0.9). The reaction mixture was worked up as quickly as possible bywashing with ice cold saturated aqueous sodium chloride (35 ml) followedby drying over sodium sulphate. The solvent was evaporated leaving anorange residue which was dissolved in dry DMF (20 ml). Without furtherpurification, sodium azide (0.226 g, 2 equiv based on quantitativetriflation) was added and the mixture stirred at room temperature undernitrogen. After 30 minutes, t.l.c. (eluant hexane:ethyl acetate, 2:1)indicated that a product had formed (R_(f) 0.4). The solvent wasevaporated, leaving a residue which was dissolved in dichloromethane (30ml) and washed with water (3×15 ml). After drying (magnesium sulphate)and evaporation of the solvent, flash chromatography (eluanthexane:ethyl acetate, 2:1) yielded7-azido-7-deoxy-2,3:5,6-di-O-isopropylidene-D-glycero-D-gulo-heptono-1,4-lactone(12) (0.42 g, 77% over two steps) as a white, crystalline solid, m.p.89°-91° C. (Found: C, 50.10; H, 6.29; N, 13.18%. C₁₃ H₁₉ N₃ O₆ requires:C, 49.84; H, 6.11; N, 13.41%); [α].sub. d²⁰ +34.57° (c, 1.00 in CHCl₃);v_(max) (CHCl₃) 2110 (N₃), 1795 (γ-lactone), 1386 and 1378 cm⁻¹ (CMe₂).

EXAMPLE 57-Azido-7-deoxy-2,3:5,6-di-O-isopropylidene-D-glycero-D-gulo-heptitol(13)

7-Azido-7-deoxy-2,3:5,6-di-O-isopropylidene-D-glycero-D-gulo-heptono-1,4-lactone(12) (1.84 g, 5.88 mmol) was dissolved in ethanol (100 ml) and stirredat 0° C. under nitrogen. Sodium borohydride (0.445 g, 2 equiv) was addedand the reaction mixture allowed to warm up to room temperature. After18 hours, t.l.c. (eluant hexane:ethyl acetate, 2:1) indicated that allstarting material had been converted to product (R_(f) 0.2). Thereaction was quenched by addition of excess solid ammonium chloride,with effervescence. Filtration and evaporation of the solvent gave aresidue which was purified by flash chromatography (eluant hexane:ethylacetate, 2:1) yielding7-azido-7-deoxy-2,3:5,6-di-O-isopropylidene-D-glycero-D-gulo-heptitol(13) (1.74 g, 93%) as a colorless, viscous oil (Found: C, 49.26; H,7.30; N, 13.26%. C₁₃ H₂₃ N₃ O₆ requires: C, 49.20; H, 7.30; N, 13.24%);[α]_(D) ²⁰ +2.87° (c, 0.94 in CHCl₃); v_(max) 3553 (broad, OH), 2107(N₃), 1384 and 1375 cm⁻¹ (CMe₂).

EXAMPLE 67-Azido-7-deoxy-2,3:5,6-di-O-isopropylidene-1,4-di-O-methanesulphonyl-D-glycero-D-gulo-heptitol(14)

7-Azido-7-deoxy-2,3:5,6-di-O-isopropylidene-D-glycero-D-gulo-heptitol(13) (0.95 g, 3.00 mmol) and 4-dimethylaminopyridine (DMAP) (1 mg) weredissolved in dry pyridine (15 ml) and stirred at 0° C. under nitrogen.Methanesulphonyl chloride (1.39 ml, 6 equiv) was added slowly and after4 hours the reaction mixture was allowed to warm up to room temperature.After 18 hours, t.l.c. (eluant hexane:ethyl acetate, 2:1) indicated thatno starting material remained (R_(f) 0.2) while a major product hadformed (R_(f) 0.25). The solvent was evaporated, leaving a red oil whichwas dissolved in ethyl acetate (150 ml) and washed with water (75 ml).After drying (magnesium sulphate) the crude mixture was purified byflash chromatography (eluant hexane:ethyl acetate, 2:1) yielding7-azido-7-deoxy-2,3:5,6-di-O-isopropylidene-1,4-di-O-methanesulphonyl-D-glycero-D-gulo-heptitol(14) (1.33 g, 94%) as a colorless, viscous oil, [α]_(D) ²⁰ +8.22° (c,1.07 in CHCl₃); v.sub. max (CHCl₃) 2109 cm⁻¹ (N₃).

EXAMPLE 71α,2α,6α,7α,7αβ-1,2:6,7-Di-O-isopropylidene-1,2,6-7-tetrahydroxypyrrolizidine (15)

7-Azido-7-deoxy-2,3:5,6-di-O-isopropylidene-1,4-di-O-methanesulphonyl-D-glycero-D-gulo-heptitol(14) (0.64 g, 1.35 mmol) was dissolved in ethanol (50 ml) and palladiumblack (10%) was added. After degassing the solution, the reactionmixture was stirred vigorously under hydrogen at room temperature fortwo hours. At this stage, t.l.c. (eluant hexane:ethyl acetate, 2:1)indicated that all starting material (R_(f) 0.25) had reacted to give aproduct which remained at the baseline. The reaction mixture wasfiltered through celite to remove the catalyst, sodium acetate (0.33 g,3 equiv based on quantitative reduction) added and the mixture stirredat 50° C. under nitrogen. After 12 hours, t.l.c. (eluant ethylacetate:methanol, 9:1) showed that the reaction mixture waspredominantly one compound (R_(f) 0.5). After evaporating the solvent,the crude mixture was purified by flash chromatography (eluant ethylacetate, increasing polarity to ethyl acetate:methanol, 9:1) giving1α,2α,6α,7α,7αβ-1,2:6,7-di-O-isopropylidene-1,2,6,7-tetrahydroxypyrrolizidine (15) (0.26 g, 76% over two steps) as a pale brown solid,m.p. 66°-69° C. (diethyl ether) (Found: C, 60.81; H, 8.44; N, 5.23%. C₁₃H₂₁ NO₄ requires: C, 61.16; H, 8.29; N, 5.49%); [α]_(D) ²⁰ +1.06° (c,1.14 in CHCl₃).

EXAMPLE 8 1α,2α,6α,7α,7αβ-1,2,6,7-Tetrahydroxy Pyrrolizidine (1)

1α,2α,6α,7α,7αβ-1,2,6,7-Di-O-isopropylidene-1,2,6,7-tetrahydroxypyrrolizidine (15) (112 mg, 0.44 mmol) was dissolved in 50% aqueoustrifluoroacetic acid (20 ml) and stirred at room temperature for sixhours. After evaporation of the solvent, the residue was dissolved inwater and purified on an ion exchange column (H⁺ form), eluting with0.5M aqueous ammonia. Freeze drying yielded1α,2α,6α,7α,7αβ-1,2,6,7-tetrahydroxy pyrrolizidine (1) (69 mg, 90%) as apale brown solid, m.p. 170°-175° C. (dec.) (Found: C, 47.62; H, 7.65; N,7.77%. C₇ H₁₃ NO₄ requires: C, 47.99; H, 7.48; N, 8.00%); [α]_(D) ²⁰ 0°(c, 1.06 in H₂ O); v_(max) (KBr disc) 3400 cm⁻¹ (very broad, OH).

EXAMPLE 97-O-tert-Butyldiphenylsilyl-2,3:5,6-di-O-isopropylidene-D-glycero-D-gulo-heptitol(16)

7-O-tert-Butyldiphenylsilyl-1,2:5,6-di-O-isopropylidene-D-glycero-D-gulo-heptono-1,4-lactone(9) (116 mg, 0.22 mmol) was dissolved in dry THF (10 ml) and stirred at0° C. under nitrogen. Lithium aluminum hydride (25 mg, 3 equiv) wasadded and the reaction mixture allowed to warm up slowly to roomtemperature. After 9 hours, t.l.c. (eluant hexane:ethyl acetate, 2:1)indicated that no starting material remained (R_(f) 0.9) while a majorproduct had formed (R_(f) 0.1). The reaction was quenched by theaddition of excess solid ammonium chloride, the mixture filtered and thesolvent evaporated. Purification by flash chromatography (eluanthexane:ethyl acetate, 3:1) yielded7-O-tert-butyldiphenylsilyl-2,3:5,6-di-O-isopropylidene-D-glycero-D-gulo-heptitol(16) (78 mg, 77%) as a colorless, viscous oil, [α]_(D) ²⁰ -2.39° (c,1.05 in CHCl₃); v_(max) (CHCl₃) 3561 (broad, OH), 1383 and 1374 cm⁻¹(CMe₂).

EXAMPLE 107-O-tert-Butyldiphenylsilyl-2,3:5,6-di-O-isopropylidene-1,4-di-O-methanesulphonyl-D-glycero-D-gulo-heptitol(17) and1,4-anhydro-7-O-tert-butyldiphenylsilyl-1-deoxy-2,3:5,6-di-O-isopropylidene-D-glycero-D-gulo-heptitol(19)

7-O-tert-Butyldiphenylsilyl-2,3:5,6-di-O-isopropylidene-D-glycero-D-gulo-heptitol(16) (260 mg, 0.49 mmol) and DMAP (1 mg) were dissolved in dry pyridine(10 ml) and stirred at 0° C. under nitrogen. Methanesulphonyl chloride(0.15 ml, 4 equiv) was added slowly and after 3 hours the reactionmixture was allowed to warm up to room temperature. After 20 hours,t.l.c. (eluant hexane:ethyl acetate, 3:2) indicated that two productshad formed (R_(f) 0.5 and 0.8) while no starting material remained(R_(f) 0.4). After evaporation of the solvent, the residue was shakenwith ethyl acetate (60 ml), leaving an insoluble brown solid. Thefiltrate was washed with water (70 ml) and dried (magnesium sulphate).After filtration and evaporation of the solvent, flash chromatography(eluant hexane:ethyl acetate, 3:1) yielded7-O-tert-butyldiphenylsilyl-2,3:5,6-di-O-isopropylidene-1,4-di-O-methanesulphonyl-D-glycero-D-gulo-heptitol(17) (224 mg, 67%) as a colorless, viscous oil, [α]_(D) ²⁰ -9.40° (c,1.08 in CHCl₃ ); and1,4-anhydro-7-O-tert-butyldiphenylsilyl-1-deoxy-2,3:5,6-di-O-isopropylidene-D-glycero-D-gulo-heptitol(19) (81 mg, 32%) as a colorless, viscous oil, [α]_(D) ²⁰ +34.71° (c,1.02 in CHCl₃); v_(max) (CHCl₃) 1382 and 1375 cm⁻¹ (CMe₂).

EXAMPLE 11N-Benzyl-7-O-tert-butylphenylsilyl-1,4-dideoxy-2,3:5,6-di-O-isopropylidene-1,4-imino-D-glycero-D-allo-heptitol(20)

7-O-tert-Butyldiphenylsilyl-2,3:5,6-di-O-isopropylidene-1,4-di-O-methanesulphonyl-D-glycero-D-gulo-heptitol(17) (147 mg, 0.21 mmol) was dissolved in benzylamine (10 ml) andstirred at 50° C. under nitrogen for 72 hours. At this stage, t.l.c.(eluant hexane:ethyl acetate, 3:1) indicated that no starting materialremained (R_(f) 0.2) while a major product had formed (R_(f) 0.8). Thebenzylamine was evaporated, leaving a dark red oil which was dissolvedin ethyl acetate (20 ml). Silica gel was added and the solventevaporated to pre-absorb the compound. Flash chromatography (eluanthexane, increasing polarity to hexane:ethyl acetate, 6:1) yieldedN-benzyl-7-O-tert-butyldiphenylsilyl-1,4-dideoxy-2,3:5,6-di-O-isopropylidene-1,4-imino-D-glycero-D-allo-heptitol(20) (94 mg, 72%) as a pale yellow, viscous oil, [α]_(D) ²⁰ -14.08° (c,1.20 in CHCl₃); v_(max) (CHCl₃) 1383 and 1375 cm⁻¹ (CMe₂).

EXAMPLE 12N-Benzyl-1,4-dideoxy-2,3:5,6-di-O-isopropylidene-1,4-imino-D-glycero-D-allo-heptitol(21)

N-Benzyl-7-O-tert-butyldiphenylsilyl-1,4-dideoxy-2,3:5,6-di-O-isopropylidene-1,4-imino-D-glycero-D-allo-heptitol(20) (94 mg, 0.16 mmol) was dissolved in dry THF (10 ml) and stirred atroom temperature under nitrogen. Tetra-n-butylammonium fluoride (0.23 mlof a 1M solution in THF, 1.5 equiv) was added and after 3 hours, t.l.c.(eluant hexane:ethyl acetate, 3:1) indicated that no starting materialremained (R_(f) 0.8) while a major product had formed (R_(f) 0.25).Evaporation of the solvent followed by flash chromatography (eluanthexane:ethyl acetate, 3:1) yieldingN-benzyl-1,4-dideoxy-2,3:5,6-di-O-isopropylidene-1,4-imino-D-glycero-D-allo-heptitol(21) (48 mg, 84%) as a colorless, viscous oil, [α]_(D) ²⁰ -58.44° (c,1.03 in CHCl₃); v_(max) (CHCl₃) 3670 (OH), 1386 and 1377 cm⁻¹ (CMe₂).

EXAMPLE 131α,2α,6α,7α,7αβ-1,2:6,7-Di-O-isopropylidene-1,2,6,7-tetrahydroxypyrrolizidine (15)

N-Benzyl-1,4-dideoxy-2,3:5,6-di-O-isopropylidene-1,4-imino-D-glycero-D-allo-heptitol(21) (91 mg, 0.25 mmol) was dissolved in dry dichloromethane (15 ml).Dry pyridine (0.04 ml, 2 equiv) was added and the solution stirred at 0°C. under nitrogen. Methanesulphonyl chloride (0.03 ml, 1.5 equiv) wasadded slowly, and after 4 hours the reaction mixture was allowed to warmup to room temperature. After 24 hours, t.l.c. (eluant hexane:ethylacetate, 3:1) indicated a product at the baseline but no startingmaterial (R_(f) 0.25). Evaporation of the solvent and trituration withdiethyl ether (2×5 ml) gave a white solid residue which was dissolved inethanol (5 ml) and added to a mixture of pre-reduced palladium black(10%) in degassed ethanol (10 ml). The resultant mixture was stirredvigorously at room temperature under hydrogen for 24 hours and thenfiltered through celite. Evaporation of the solvent gave a white solidresidue which was dissolved in ethyl acetate (20 ml), washed withsaturated aqueous sodium hydrogen carbonate (10 ml) and dried (magnesiumsulphate). Flash chromatography (eluant ethyl acetate, increasingpolarity to ethyl acetate:methanol, 9:1) yielded1α,2α,6α,7α,7αβ-1,2:6,7-di-O-isopropylidene-1,2,6,7-tetrahydroxypyrrolizidine (15) (20 mg, 31%) as a pale yellow oil with spectroscopicdata identical to those in Example 7, above.

Various other examples will be apparent to the person skilled in the artafter reading the present disclosure without departing from the spiritand scope of the invention. It is intended that all such other examplesbe included within the scope of the appended claims.

What is claimed is: 1.1α,2.alpha.,6α,7α,7αβ-1,2,6,7-tetrahydroxypyrrolizidine.
 2. The methodof inhibiting a glycosidase enzyme in a biological fluid containing saidenzyme comprising subjecting said fluid to an effective amount of thecompound of claim 1 suitable to inhibit said glycosidase. 3.7-O-tert-Butyldiphenylsilyl-2,3:5,6-di-O-isopropylidene-D-glycero-D-gulo-heptono-1,4-lactone.4.1α,2α,6α,7α,7αβ-1,2:6,7-di-O-isopropylidene-1,2,6,7-tetrahydroxypyrrolizidine.